Depletion of RNASEH2 Activity Leads to Accumulation of DNA Double-strand Breaks and Reduced Cellular Survivability in T Cell Leukemia.

Ribonuclease H2 (RNase H2) is a member of the ribonuclease H family of enzymes involved in removal of RNA from RNA-DNA hybrids as well as ribonucleotides which get misincorporated into the genomic DNA. Recent studies have shown that RNase H2 function is also needed for successful DNA repair through NHEJ events where DNA pol µ uses ribonucleotides during the gap filling stage. Mammalian RNase H2 is composed of three subunits, RNASEH2A, RNASEH2B and RNASEH2C. There have been studies suggesting changes in expression of these genes in various cancers of breast, prostate, colon, liver, and kidney. In this study, we have investigated the functional role of RNASEH2A and RNASEH2B in leukemic T-cells, MOLT4 and Jurkat. shRNA mediated knockdown of RNASEH2A/ RNASEH2B expression led to reduced cell survival and increase in apoptotic cell population. Importantly, knockdown of RNASEH2A or RNASEH2B, led to cell cycle arrest at S phase and increased number of 53BP1 foci due to abrogation of NHEJ. Interestingly, RNASEH2A or RNASEH2B depleted cells showed significantly retarded DSB repair kinetics compared to scrambled shRNA control, when exposed to ionizing radiation suggesting that NHEJ is abrogated due to loss of RNASEH2 activity in T-ALL cells. Thus, we uncover the importance of RNase H2 function in leukemic cells and suggest that it can be targeted for cancer therapy.

TonEBP recognizes R-loops and initiates m6A RNA methylation for R-loop resolution.

R-loops are three-stranded, RNA-DNA hybrid, nucleic acid structures produced due to inappropriate processing of newly transcribed RNA or transcription-replication collision (TRC). Although R-loops are important for many cellular processes, their accumulation causes genomic instability and malignant diseases, so these structures are tightly regulated. It was recently reported that R-loop accumulation is resolved by methyltransferase-like 3 (METTL3)-mediated m6A RNA methylation under physiological conditions. However, it remains unclear how R-loops in the genome are recognized and induce resolution signals. Here, we demonstrate that tonicity-responsive enhancer binding protein (TonEBP) recognizes R-loops generated by DNA damaging agents such as ultraviolet (UV) or camptothecin (CPT). Single-molecule imaging and biochemical assays reveal that TonEBP preferentially binds a R-loop via both 3D collision and 1D diffusion along DNA in vitro. In addition, we find that TonEBP recruits METTL3 to R-loops through the Rel homology domain (RHD) for m6A RNA methylation. We also show that TonEBP recruits RNaseH1 to R-loops through a METTL3 interaction. Consistent with this, TonEBP or METTL3 depletion increases R-loops and reduces cell survival in the presence of UV or CPT. Collectively, our results reveal an R-loop resolution pathway by TonEBP and m6A RNA methylation by METTL3 and provide new insights into R-loop resolution processes.

Structural and inhibition studies of the RNase H function of xenotropic murine leukemia virus-related virus reverse transcriptase.

RNase H inhibitors (RNHIs) have gained attention as potential HIV-1 therapeutics. Although several RNHIs have been studied in the context of HIV-1 reverse transcriptase (RT) RNase H, there is no information on inhibitors that might affect the RNase H activity of other RTs. We performed biochemical, virological, crystallographic, and molecular modeling studies to compare the RNase H function and inhibition profiles of the gammaretroviral xenotropic murine leukemia virus-related virus (XMRV) and Moloney murine leukemia virus (MoMLV) RTs to those of HIV-1 RT. The RNase H activity of XMRV RT is significantly lower than that of HIV-1 RT and comparable to that of MoMLV RT. XMRV and MoMLV, but not HIV-1 RT, had optimal RNase H activities in the presence of Mn²âº and not Mg²âº. Using hydroxyl-radical footprinting assays, we demonstrated that the distance between the polymerase and RNase H domains in the MoMLV and XMRV RTs is longer than that in the HIV-1 RT by ∼3.4 Å. We identified one naphthyridinone and one hydroxyisoquinolinedione as potent inhibitors of HIV-1 and XMRV RT RNases H with 50% inhibitory concentrations ranging from ∼0.8 to 0.02 µM. Two acylhydrazones effective against HIV-1 RT RNase H were less potent against the XMRV enzyme. We also solved the crystal structure of an XMRV RNase H fragment at high resolution (1.5 Å) and determined the molecular details of the XMRV RNase H active site, thus providing a framework that would be useful for the design of antivirals that target RNase H.

Determination of the role of the human RNase H1 in the pharmacology of DNA-like antisense drugs.

Although ribonuclease H activity has long been implicated as a molecular mechanism by which DNA-like oligonucleotides induce degradation of target RNAs, definitive proof that one or more RNase H is responsible is lacking. To date, two RNase H enzymes (H1 and H2) have been cloned and shown to be expressed in human cells and tissues. To determine the role of RNase H1 in the mechanism of action of DNA-like antisense drugs, we varied the levels of the enzyme in human cells and mouse liver and determined the correlation of those levels with the effects of a number of DNA-like antisense drugs. Our results demonstrate that in human cells RNase H1 is responsible for most of the activity of DNA-like antisense drugs. Further, we show that there are several additional previously undescribed RNases H in human cells that may participate in the effects of DNA-like antisense oligonucleotides.

The protein components and mechanism of eukaryotic Okazaki fragment maturation.

An initiator RNA (iRNA) is required to prime cellular DNA synthesis. The structure of double-stranded DNA allows the synthesis of one strand to be continuous but the other must be generated discontinuously. Frequent priming of the discontinuous strand results in the formation of many small segments, designated Okazaki fragments. These short pieces need to be processed and joined to form an intact DNA strand. Our knowledge of the mechanism of iRNA removal is still evolving. Early reconstituted systems suggesting that the removal of iRNA requires sequential action of RNase H and flap endonuclease 1 (FEN1) led to the RNase H/FEN1 model. However, genetic analyses implied that Dna2p, an essential helicase/nuclease, is required. Subsequent biochemical studies suggested sequential action of RPA, Dna2p, and FEN1 for iRNA removal, leading to the second model, the Dna2p/RPA/FEN1 model. Studies of strand-displacement synthesis by polymerase delta indicated that in a reconstituted system, FEN1 could act as soon as short flaps are created, giving rise to a third model, the FEN1-only model. Each of the three pathways is supported by different genetic and biochemical results. Properties of the major protein components in this process will be discussed, and the validity of each model as a true representation of Okazaki fragment processing will be critically evaluated in this review.

Expression of Moloney murine leukemia virus RNase H rescues the growth defect of an Escherichia coli mutant.

A 157-amino-acid fragment of Moloney murine leukemia virus reverse transcriptase encoding RNase H is shown to rescue the growth-defective phenotype of an Escherichia coli mutant. In vitro assays of the recombinant wild-type protein purified from the conditionally defective mutant confirm that it is catalytically active. Mutagenesis of one of the presumptive RNase H-catalytic residues results in production of a protein variant incapable of rescue and which lacks activity in vitro. Analyses of additional active site mutants demonstrate that their encoded variant proteins lack robust activity yet are able to rescue the bacterial mutant. These results suggest that genetic complementation may be useful for in vivo screening of mutant viral RNase H gene fragments and in evaluating their function under conditions that more closely mimic physiological conditions. The rescue system may also be useful in verifying the functional outcomes of mutations based on protein structural predictions and modeling.

Mutating a conserved motif of the HIV-1 reverse transcriptase palm subdomain alters primer utilization.

In order to investigate how primer grip residues of human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) contribute toward the architecture of its palm subdomain and neighboring structural elements, the DNA polymerase and ribonuclease H (RNase H) activities of enzymes bearing aromatic substitutions at Trp229 and Tyr232 of the catalytically-competent p66 subunit were evaluated. Although all mutants retained RNase H function, the manner in which different RNA-DNA hybrids were hydrolyzed was affected. Depending on the nature of the substitution, DNA-dependent DNA synthesis was (i) unaffected, (ii) interrupted shortly after initiation, or (iii) stalled when the replication machinery encountered an intramolecular duplex on the single-stranded template. Evaluating (-) strand strong-stop DNA synthesis on an RNA template derived from the viral genome raises the additional possibility that DNA and RNA primers might be differentially recognized by the retroviral polymerase. In support of this, all mutants were unable to extend the HIV-1 polypurine tract (PPT) RNA primer into (+) strand DNA, despite supporting the equivalent event from an oligodeoxynucleotide primer. Collectively, our data illustrate that subtle alterations to primer grip architecture may manifest themselves in discrimination between oligoribo- and oligodeoxyribonucleic acid primers.

RNase H-independent antisense activity of oligonucleotide N3 '--> P5 ' phosphoramidates.

Oligonucleotide N3'-->P5'phosphoramidates are a new and promising class of antisense agents. Here we report biological properties of phosphoramidate oligonucleotides targeted against the human T cell leukemia virus type-I Tax protein, the major transcriptional transactivator of this human retrovirus. Isosequential phosphorothioate oligodeoxynucleotides and uniformly modified and chimeric phosphoramidate oligodeoxynucleotides containing six central phosphodiester linkages are all quite stable in cell nuclei. The uniformly modified anti-tax phosphoramidate oligodeoxynucleotide does not activate nuclear RNase H, as was shown by RNase protection assay. In contrast, the chimeric phosphoramidate-phosphodiester oligodeoxynucleotide is an efficient activator of RNase H. The presence of one or two mismatched nucleotides in the phosphodiester portion of oligonucleotides affected this activation only negligibly. When introduced into tax-transformed fibroblasts ex vivo, only the uniformly modified anti-tax phosphoramidate oligodeoxynucleotide caused a sequence-dependent reduction in the Tax protein level. Neither the chimeric phosphoramidate nor the phosphorothioate oligodeoxynucleotides significantly reduced tax expression under similar experimental conditions.

Inhibition of expression of the multidrug resistance-associated P-glycoprotein of by phosphorothioate and 5' cholesterol-conjugated phosphorothioate antisense oligonucleotides.

Multiple drug resistance (MDR) as a result of overexpression of the P-glycoprotein drug transporter, a product of the MDR1 gene, is a significant problem in cancer therapeutics. We demonstrate that phosphorothioate antisense oligonucleotides can reduce levels of MDR1 message, inhibit expression of P-glyco protein, and affect drug uptake in MDR mouse 3T3 fibroblasts. An obligonucleotide (5995) directed against a sequence overlapping the AUG start codon was effective in reduction MDR1 transcript and protein levels when used at submicromolar concentrations in conjunction with cationic liposomes, whereas a scrambled control oligonucleotide (10221) was ineffective. Substantial and specific antisense effects could also be attained with a 5' cholesterol conjugate of the 5995 sequence. In this case, use of cationic liposomes was unnecessary. The 5' cholesterol 5995, but the not 5' cholesterol 10221, reduced MDR1 message and P-glycoprotein levels by 50-60% when used at low micromolar concentrations. In parallel, treatment with 5' cholesterol 5995 also enhanced cellular accumulation of rhodamine 123, a well-known substrate of the P-glycoprotein transporter. The effectiveness of the cholesterol-conjugated 5995 may be due to its rapid and extensive cell uptake, as indicated in flow cytometry and confocal microscopy studies. These observations suggest that cholesterol-conjugated anti-sense oligonucleotides may offer a novel approach to inhibition of P-glycoprotein-mediated MDR and to the modulation of other tumor cell genes whose overexpression contributes to the neoplastic state or to resistance to therapy.

Effects on DNA synthesis and translocation caused by mutations in the RNase H domain of Moloney murine leukemia virus reverse transcriptase.

To determine the various roles of RNase H in reverse transcription, we generated a panel of mutations in the RNase H domain of Moloney murine leukemia virus reverse transcriptase based on sequence alignments and the crystal structures of Escherichia coli and human immunodeficiency virus type 1 RNases H (S. W. Blain and S. P. Goff, J. Biol. Chem. 268:23585-23592, 1993). These mutations were introduced into a full-length provirus, and the resulting genomes were tested for infectivity by transient transfection assays or after generation of stable producer lines. Several of the mutant viruses replicated normally, some showed significant delays in infectivity, and others were noninfectious. Virions were collected, and the products of the endogenous reverse transcription reaction were examined to determine which steps might be affected by these mutations. Some mutants left their minus-strand strong-stop DNA in RNA-DNA hybrid form, in a manner similar to that of RNase H null mutants. Some mutants showed increased polymerase pausing. Others were impaired in first-strand translocation, independently of their wild-type ability to degrade genomic RNA, suggesting a new role for RNase H in strand transfer. DNA products synthesized in vivo by the wild-type and mutant viruses were also examined. Whereas wild-type virus did not accumulate detectable levels of minus-strand strong-stop DNA, several mutants were blocked in translocation and did accumulate this intermediate. These results suggest that in vivo wild-type virus normally translocates minus-strand strong-stop DNA efficiently.

Mutating the "primer grip" of p66 HIV-1 reverse transcriptase implicates tryptophan-229 in template-primer utilization.

"BcgI cassette" mutagenesis was used to prepare variants of p66 human immunodeficiency virus (HIV)-1 reverse transcriptase with amino acid substitutions between residues Glu224 and Trp229. Mutant polypeptides were reconstituted in vitro with wild type p51 to generate the "selectively mutated" heterodimer series p66(224A)/p51-p66(229A)/p51. Purified enzymes were characterized with respect to dimerization, DNA polymerase, RNase H, and tRNA(Lys-3) binding. The combined analyses indicate that while alteration of p66 residues Glu224-Leu228 has minimal consequences, the DNA polymerase activities of mutant p66(229A)/p51 are impaired. DNase I footprinting illustrates that this mutant does not form a stable replication complex with a model template-primer. In vivo studies indicate that the equivalent mutation eliminates viral infectivity, suggesting a contribution of Trp229 toward architecture of the p66 primer grip.

Antisense inhibition of oncogene expression.

To understand the role of individual genes in regulating biological processes, one must be able to interfere specifically with either their expression or function. While monoclonal antibodies have proven very useful in studying cell surface proteins, the specific inhibition of intracellular proteins in viable cells is a much more difficult problem. The goal of antisense technology is to develop small oligonucleotides, plasmids, or retroviral vectors which can be readily introduced into living cells in order to inhibit specific gene expression. In this review, we briefly describe the principles of antisense usage, including problems of cellular uptake and intracellular distribution, mechanism of antisense action, and the properties of various oligonucleotide derivatives. In addition we present several examples of the biological effects of antisense administration used to study the role of specific genes in the regulation of cell growth and differentiation.